Clostridium difficile is the most common cause of hospital-acquired infectious diarrhea, at a cost of greater than 1.1 billion dollars per year in the United States alone. Despite the clinical impact of C. difficile it remains unclear how C. difficile colonizes the host and evades the host immune defenses. Several lines of evidence suggest antimicrobial peptides of the innate immune system play an important role in controlling C. difficile infections. However how C. difficile responds to the stress caused by antimicrobial peptides is poorly understood. One major reason for this lack of understanding is the inability to genetically manipulate this strict anaerobe. We have successfully constructed stable targeted mutations in C. difficile. Our long-term goal is to better understand how C. difficile resists the innate immune defenses during an infection. Extra-Cytoplasmic Function (ECF) factors represent an important class of signal transduction systems which respond to cell envelope stresses. Although ECF factors are involved in resistance to innate immune defenses in Gram- negative pathogens, the role of ECF factors during infections caused by Gram-positive pathogens has not been established. The objective of this application is to define the role of ECF sigma factors in the cell envelope stress response of C. difficile and determine to how these ECF sigma factors contribute to the virulence properties of C. difficile. Preliminary evidence suggests the C. difficile genome encodes three ECF sigma factors which are induced in response to cell envelope stress. The role of ECF sigma factors in resistance cell envelope stress and pathogenesis C. difficile will be determined in the following specific aims: 1) Determine how PrsW controls expression of the C. difficile ECF factors CsfT and CsfU, 2) Define the role of these C. difficile ECF factors in resistance to cell envelope stress and pathogenesis, and 3) Identify the regulons of these C. difficile ECF factors. The proposed research is relevant to the mission of the NIH because it is expected to advance our knowledge of how this increasingly clinically important Gram-positive pathogen resists cell envelope stress and host innate immune defenses. A better understanding of the cell envelope stress response may provide new targets for treatment of C. difficile infections.